Ribonucleotide reductases (RNRs) catalyze an essential step in DNA replication and repair: the conversion of purine and pyrimidine nucleotides to deoxynucleotides. Their central role in nucleic acid metabolism has made them the successful target of the antitumor agents gemzar and hydroxyurea. The RNRs have been divided into classes based on the unusual metallo-cofactors required to generate a transient thiyl radical that initiates radical dependent nucleotide reduction. Presteady state experiments using rapid chemical quench and HPLC analysis, rapid freeze quench and EPR analysis and stopped flow visible and fluorescence spectroscopies will be carried out to complete mechanistic studies on class I (diiron-tyrosyl radical (Y.)) and class II (adenosylcobalamin) RN As. The mechanism of thiyl radical formation on one subunit (R1) by the diiron-Y. on the second subunit (R2) of the class I RNRs over 35 A by long range electron coupled proton transfer will be examined using semisynthetic proteins and peptides which can initiate reduction by laser flash photolysis. The mechanism by which di- and tri-phosphates of gemzar inactivate class I and II RNRs respectively will be examined. Regulation of all RNRs is multilayered. Allosteric regulation governs their substrate specificity and substrate turnover. Regulation at the transcriptional level maximizes RNR activity in the S phase of the cell cycle. Regulation can also occur at the translational level, protein degradation level and by phosphorylation. Addition of replication blocks or DNA damaging agents to cells has revealed the central role of RNRs in cell surveillance. There are two class I RNRs in yeast. The availability of mRNA transcriptional profiling, knockouts of every gene in isogenic strains, the ease with which genes can be replaced by homologous recombination, and the ability to carry out biochemistry, have made yeast our organism of choice to study regulation. Our efforts are focused on understanding the active subunit composition in yeast, subunit location in the cell and how the layers of regulation interact. The mechanism by which the diiron-Y. cofactor is assembled in vivo is also being investigated. Understanding the regulation in yeast, with its many counterparts in humans, should lead to design of more effective therapeutics.